The invention relates to a process for the preparation of 5,10-methenyl-(6R)-tetrahydrofolic acid [5,10-CH-(6R)-THF; "(6R)-I"], of biologically active tetrahydrofolates, especially of 5-formyl-[5-CHO-(6S)-THF] and 5-methyl-(6S)-tetrahydrofolic acid [5-Me-(6S)-THF] as well as of the salts thereof, especially the physiologically well tolerated calcium, magnesium, sodium and potassium salts thereof.
The said (6S)-tetrahydrofolates [(6S)-5,6,7,8-tetrahydropteroyl-L-glutamates] are the sufficiently stable natural biologically active forms of folic acid (folic acid cofactors).
5-CHO-(6S)-THF is the citrovorum factor (=growth factor for Leuconostoc citrovorum).
In the organism 5-CHO-(6S)-THF is converted into 5-Me-(6S)-THF via 5,10-methylene-(6R)-THF.
Tetrahydrofolates contain 2 asymmetric centers. When they are synthesized from folic acid [N-(pteroyl)-L-glutamic acid] the chiral C atom contained in the glutamic acid residue is in the L form, whereas the chiral C atom produced in position 6 [C(6)] by hydrogenation of the double bond in the 5,6 position of the pteroyl radical is present in the racemic, the (6R,S), form. Accordingly, all synthetic tetrahydrofolates consist of a 1:1 mixture of two diastereomers.
The tetrahydrofolates which occur naturally, for example in the liver, are found only in one diastereomeric form, with 5-CHO-THF being in the form of 5-CHO-(6S)-THF, and 5-Me-THF being in the form of 5-Me-(6S)-THF.
The absolute configuration at C(6) of natural tetrahydrofolic acid is, according to J. C. Fontecilla-Camps et al., J. Amer. Chem. Soc. 101 (20), 6114/5 (1979), in accordance with the sequence rule to be specified as S, and that at C (6) of natural 5,10-methylene-tetrahydrofolic acid and of 5,10-methenyl-tetrahydrofolic acid to be specified as R: R. Kalbermatten et al., Helv. chim. Acta 64 (8), 2627 (1981), footnote 4.
5-CHO-(6R,S)-THF (folinic acid) is used in the form of its calcium salt (leucovorin) as a pharmaceutical for the treatment of megaloblastic folate-deficiency anemia, as an antidote to increase the tolerability of folic acid antagonists, specifically of aminopterin, methotrexate and fluorouracil in cancer therapy ("leucovorin rescue") and the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis, as well as to increase the tolerability of certain antiparasitics, for example trimethoprim-sulfamethoxazole, in chemotherapy.
Calcium 5-methyl-(6R,S)-tetrahydrofolate is used similarly.
Administration of 5-CHO-(6R,S)-THF is followed by rapid conversion of the (6S) portion of this diastereomeric mixture into 5-Me-(6S)-THF, whereas the (6R) portion is not metabolized: J. A. Straw et al., Cancer Research 44, 3114-3119 (1984).
In addition, 5-CHO-(6R)-THF inhibits some of the enzymes responsible for C.sub.1 transfer and thus the biochemical action of the tetrahydrofolates: R. P. Leary et al., Biochem. Biophys. Res. Commun. 56, 484 (1973); V. F. Scott et al., ibid. 14, 523 (1964); G. K. Smith et al., Biochemistry, 20, 4034 (1981). Hence the use of (6S)-tetrahydrofolates in place of (6R,S)-tetrahydrofolates ought also to have therapeutic advantages.
There is therefore a need to replace the mixtures of diastereomers, containing the (6S) and (6R) forms in each case, which have hitherto been used, with the natural (6S) form.
Many attempts have been made to resolve (6R,S)-tetrahydrofolic acids and to carry out the asymmetric synthesis thereof and to isolate the physiologically active forms.
D. Cosulich et al., J. Amer. Chem. Soc. 74, 4215-16 (1952), U.S. Pat. No. 2,688,018 (Aug. 31, 1954), have attempted, for example, to bring about the resolution by fractional crystallization of an alkaline earth metal salt, for example the calcium or strontium salt, of 5-CHO-(6R,S)-THF from an aqueous solution [see also J. C. Fontecilla-Camps et al., J. Amer. Chem. Soc. 101, 6114 (1979)].
The desired resolution cannot be achieved under the conditions published by B. Cosulich et al. On crystallization of, for example, the calcium salt of 5-CHO-(6R,S)-THF from water at pH 7-8 it is always the 6R,S form which is recovered, as can be demonstrated quantitatively by means of chromatographic analysis on a chiral HPLC column as well as on the basis of the optical rotation. It is immaterial in this connection whether crude or pure calcium salt of 5-CHO-(6R,S)-THF is used for the crystallization; the (6R,S) form is always recovered. Nor is it possible to resolve and enrich the (6S) form by seeding a supersaturated aqueous solution of an alkaline earth metal salt of 5-CHO-(6R,S)-THF with authentic alkaline earth metal salt of 5-CHO-(6S)-THF.
Resolution of the pairs of diastereomers has also been attempted by chromatography: J. Feeney et al., Biochemistry 20, 1837 (1981). In addition the (6S) isomers have been prepared by stereospecific reduction of dihydrofolates in the presence of dihydrofolate reductase: L. Rees et al., Tetrahedron 42, 117 (1986).
L. Rees et al., J. Chem. Soc. Chem. Commun. 1987, 470 and EP-A No. 2,266,042 have described a process for the resolution of (6R,S)-THF with which it was possible to produce small amounts of 5-CHO-(6S)-THF and 5-CHO-(6R)-THF. The process comprises reacting (6R,S)-THF with, for example, (-)-menthyl chloroformate to give the diastereomeric 5-(-)-menthyloxycarbonyl-tetrahydrofolic acids, separating these by repeated treatment with n-butanol, heating the resulting diastereomers with a saturated solution of hydrogen bromide in a mixture of formic acid and acetic acid, with formation after hydrolysis of 5-formyl-(6S)-and -(6R)-THF, and finally isolating the latter as calcium salts.
This process is laborious and difficult and requires highly toxic phosgene to prepare the chiral reagent. In addition, the starting material (6R,S)-THF is very unstable. On elimination of the chiral accessory group with HBr in AcOH at &gt;50.degree. C. there is partial elimination of the glutamic acid, and by-products which can be removed only with difficulty are formed. The (6S)-folinic acid produced by a process of this type would be so costly that it would be scarcely possible to use it in place of (R,S)-tetrahydrofolates.
No industrially utilizable process for obtaining (6S)-tetrahydrofolates has hitherto been disclosed. Thus, the object was still to find a straightforward and industrially applicable process for the preparation of 5-CHO-and 5-Me-(6S)-THF.
5-CHO- and 5-Me-(6R,S)-THF can be prepared in a known manner from folic acid. Formylation with formic acid produces 10-formyl-folic acid (10-CHO-FA). The latter can subsequently be catalytically hydrogenated to 10-formyl-tetrahydrofolic acid (10-CHO-THF). 5,10-Methenyl-tetrahydrofolic acid (I; 5,10-CH-THF; "anhydroleucovorin") can be obtained from the latter by dehydration: ##STR1##
In this formula, X.sup..crclbar. is one equivalent of any desired anion, for example Cl.sup..crclbar. or Br.sup..crclbar.. I can also be in the form of an acid addition salt, for example the chloride hydrochloride of the abbreviated formula (5,10-CH-THF).sup..sym. Cl.sup..crclbar..HCl. The corresponding inner salt (I, X.sup..crclbar. absent, COO.sup..crclbar. in place of COOH) can also be obtained from the latter form with the aid of an anion exchanger.
The desired tetrahydrofolates can easily be prepared from I: hydrolysis (transformylation) results in 5-formyl-tetrahydrofolic acid (5-CHO-THF) which can be isolated as the calcium salt (leucovorin): Swiss Patent Specification No. 305,574 (CYANAMID). Reduction with sodium borohydride results in 5-methyl-tetrahydrofolic acid (5-Me-THF; mefolinic acid) which can be isolated as the calcium or magnesium salt: Swiss Patent Specification No. 649,550 (EPROVA).